This invention is generally directed to a chip-based element for the stepwise bioselection, bioreaction, characterization, and identification of biomolecules. More specifically, the bioactive chip or probe is capable of separation of biomolecules residing in complex mixtures, post-separation processing and/or modifying, and complete structural characterization of the processed and/or modified biomolecules by mass spectrometry.
Grant Support
Financial assistance for some of the work reported herein was provided by the National Institutes of Health, under grant number 1 R43 CA82079-01. The National Institute of Health may own certain rights to this invention.
Massive, worldwide efforts over the past few decades have resulted, or will soon result, in the complete genome sequencing of a number of select organisms. Once completed and cataloged into databases, these genomes represent virtual libraries that can be translated into all of the potential proteins contained within their respective organisms. In this regard, genome databases become a foundation for the much broader field of proteomics, wherein the structure and function of specific proteins are under investigation. Such studies are intrinsically complicated, representing multi-dimensional problems that are both qualitative and quantitative in nature. To begin with, a protein of interest will generally reside in complex biological systems and oftentimes represents only a small fraction of the total protein content of the system. The efficient fractionation of the protein from bulk endogenous compounds is therefore necessary for any further characterization.
Once isolated, the protein must be characterized in terms of structure. The focus of these analyses ranges from primary (amino acid sequence) through tertiary (three-dimensional structure due to protein folding) structure, as well as post-translational modifications. Proteome investigation then takes on the new dimension of protein function, with emphasis placed on quaternary structure (polypeptide complexes resulting in functional proteins) and the determination of biomolecular interaction partners (receptor-ligand interactions).
Finally, it is often necessary to quantitatively monitor expression profiles to better understand protein function as a part of a complete cellular system.
In all, it is accurate to say that proteome investigations demand much of analytical sciences and corresponding instrumentation. Thus, there exists a growing need for concerted, multi-analytical approach capable of high-sensitivity analyte fractionation and characterization of protein structure and function.
Multidimensional chip-integrated microarrays that encompass both selective protein fractionation and complete structural/functional characterization would satisfy the need for such proteome analysis using xe2x80x9clab-on-a-chipxe2x80x9d approaches. These chip-based microarrays must be combined with a microfluidics system able to precisely deliver a sample to specialized sites on the chip that are capable of analyte fractionation and subsequent processing and/or modifications. The chip based microfluidics systems preferably includes a form of detection for sensing the isolation of an analyte and for tracking the location of the analyte on the array during manipulation. Finally, the system must be capable of providing defining structural information on the analyte, such as sequence verification and/or identification, and detection of point mutations and post-translational modification, thereby resulting in analyte identification.
In 1988, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was introduced by Hillenkamp and Karas (Anal. Chem. 60:2288-2301, 1988), and, has since become a valuable tool for protein characterization and identification. Briefly, MALDI-TOF mass spectrometry is based on the ability to generate intact vapor-phase ions of large, thermally labile biomolecules by desorption/ionization from a matrix comprised of small volatile (matrix) molecules and the biomolecules studied. Pulsed laser radiation, tuned to an absorption maximum of the matrix is used to initiate the desorption/ionization event and to simultaneously generate a packet of ions of different mass-to-charge ratio (m/z). These ions are accelerated to the same electrostatic potential and allowed to drift an equal distance before striking a detector. The mass of the ions is determined by equating the flight times of the ions to m/z.
The success of MALDI-TOF mass spectrometry in the area of protein science can be attributed to several factors. The greatest of these is that MALDI-TOF can be rapidly (xcx9c5 minutes) applied as an analytical technique to analyze small quantities of virtually any protein (practical sensitivities of xcx9c1 pmole protein loaded into the mass spectrometer). The technique is also extremely accurate in identifying biomolecules. Beavis and Chait demonstrated that the molecular masses of peptides and proteins can be determined to within 0.01% by using methods in which internal mass calibrants (x-axis calibration) are introduced into the analysis (Anal. Chem. 62:1836-40, 1990).
MALDI-TOF can also be quantitatively used with a similar method in which internal reference standards are introduced into the analysis for ion signal normalization (y-axis calibration) with accuracies on the order of 10% (Nelson et al., Anal. Chem. 66:1408-15, 1994).
Finally, MALDI-TOF is extremely tolerant of large molar excesses of buffer salts and, more importantly, the presence of other proteins, making it a practical approach for directly analyzing complex biological mixtures. Many examples exist where MALDI-TOF is used to directly analyze the results of proteolytic or chemical degradation of polypeptides. Other examples extend to elucidating post-translational modifications (namely carbohydrate type and content), a process requiring the simultaneous analysis of components present in a heterogeneous glycoprotein mixture (Sutton et al., Techniques in Protein Chemistry III, Angeletti, Ed., Academic Press, Inc., New York, pp. 109-1 16, 1993). Arguably, the most impressive use of direct mixture analysis is the screening of natural biological fluids. In this application, proteins are identified, as prepared directly from the host fluid, by detection at precise and characteristic mass-to-charge (m/z) values (Tempst et al., Mass Spectrometry in the Biological Sciences, Burlingame and Carr, Ed., Humana Press, Totowa, N.J., p. 105, 1996).
While the above examples involving direct MALDI-TOF analysis of complex mixtures are quite impressive, there exist limits to the extent of practical application. These limits are reached when a target analyte is a minor component of the mixture, and is present at low concentration. A common occurrence in such situations is that the target analyte is never observed in the MALDI-TOF mass spectrum. This lack of detection is generally due to the low concentration of the analyte, yielding ion signals at or below the instrumental limits of detection. This effect is further exacerbated by protein-analyte interactions xe2x80x9cstealingxe2x80x9d analyte molecules from the MALDI-TOF process and/or a high instrumental baseline produced from other proteins present in the mixture resulting in xe2x80x9canalyte maskingxe2x80x9d. Methods for the selective concentration of specific species in the mixture were therefore required in order to achieve ion signals from the target analyte.
Biomolecular Interaction Analysis (BIA) is a technique capable of monitoring interactions, in real time and without the use of labels, between two or more molecules such as proteins, peptides, nucleic acids, carbohydrates, lipids and low molecular weight molecules (i.e. signaling substances and pharmaceuticals). Molecules do not need to be purified or even solubilized for BIA, but can be studied in crude extracts as well as anchored in lipid vesicles, viruses, bacteria and eucaryotic cells. In one form, the detection of molecular interactions in BIA is via Surface Plasmon Resonance (SPR), taking form of SPR-BIA. The detection principle of SPR-BIA relies on the optical phenomenon of SPR, which detects changes in the refractive index of the solution close to the surface of a sensor device, or chip. An SPR sensor consists of a transparent material having a metal layer deposited thereon. One of the interactants (e.g., receptor) is immobilized on the metal surface layer of the sensor that forms one wall of a micro-action site. A light source generates polarized light that is directed through a prism, or diffraction grating, striking the metal layer-transparent material interface. A detector detects light reflected from the metal surface. A sample containing the other interaction partner is injected in a controlled flow over the surface containing the bound interactant. Any change in the surface concentrations resulting from an interaction between the two, or more, interactants, is spectroscopically detected as an SPR signal by the shifting of relative reflective intensity signals. A continuous display of the SPR signal, as a function of time, yields a xe2x80x9csensorgramxe2x80x9d that provides a complete record of the progress of associations and disassociations. When analysis of one interaction cycle is completed, the surface of the sensor can be regenerated by treatment with conditions that remove all bound analytes without affecting the activity of the immobilized ligand. SPR-BIA has become a valuable tool for the functional characterization of proteins and is broadly used for determining the kinetic and affinity parameters involved in biomolecular interactions; however, no structural information on the interacting biomolecules can be gained from the SPRIBIA analysis.
In a recent invention, SPR-BIA and MALDI-TOF MS were combined in a method that allows selective retrieval and retention of an analyte from solution on a sensor chip (monitored by SPR-BIA), followed by MALDI-TOF MS analysis of the sensor chip, yielding the mass of the retained analyte (see U.S. Pat. No. 5,955,729 entitled xe2x80x9cSurface Plasmon Resonance-Mass Spectrometry (SPR-MS)xe2x80x9d. Thereby, this method performs a selective concentration of specific analytes from the analyzed mixture in quantities sufficient to achieve analyte ion signals for MALDI-TOF MS. Whereas the SPR-MS combination represents an important improvement over the SPR-BIA analysis alone and allows for functional concentration of a targeted analyte (important for MALDI-TOF MS analysis of low-level analytes), both SPR-BIA and SPR-MS have been limited to analyzing biomolecules as they exist in the solution, which, in many instances, can not provide enough information for the biomolecules identification. Frequently, in order to fully characterize and identify certain biomolecules, modification or derivatization is required.
Thus, there is a need for devices and methods that allow for the isolation of small quantities of biomolecules from a complex solution and that are able to modify, or bioreact, these biomolecules to create characteristic fragments, derivatives and the like. Accordingly, a variety of chemistries, enzymologies, characterizations and identification techniques may be employed after analyte isolation to gain information on both original and reacted biomolecules. Importantly, these chemistries, enzymologies, characterizations and identification techniques must be performed in manners that do not introduce interfering artifacts, e.g., backgrounds arising from the modifying reagents, the immobilized receptor responsible for isolating the analyte or from instrumental changes, into the analysis. Therefore, it is preferred to perform these characterizing modifications using: immobilized reagents, such as enzymes and the like (to preclude the introduction of reagent-induced artifacts), which are present at different sites than the receptor (such as not to introduce artifacts due to the receptor), and that analyses are performed on the same platform (such as to minimize instrumental changes). The present invention fulfills these needs, and provides further related advantages
Briefly stated, the present invention is directed to a bioreactive probe or chip (BC) that allows for the isolation of analytes, such as biomolecules, followed by modification or bioreaction on these said analytes. More specifically, the present invention relates to various methods and apparatuses that include the BC and further include characterization and identification technologies, such as Bioactive Chip Mass Spectrometry (BCMS).
Within the context of the present invention, BC provides a method and device for the capture and subsequent digestion or derivatization of an analyte. Further, real-time information regarding a variety of molecular interactions may be provided by techniques such as interaction analysis (IA). Finally, the variety of molecules is localized and concentrated thereby aiding in the identification and/or quantification of the molecules by techniques such as mass spectroscopy (MS).
In one embodiment, a device for performing BCMS is disclosed. Preferably, the device consists of a chip with separate addressable sites, these sites created for the purposes of analyte separation, processing and modification.
In a related embodiment, a device in which the addressable sites present on the chip are brought into fluid communication with each other is disclosed. Preferably, the chip has separate addressable sites for the purposes of analyte separation, processing and modification is used in conjunction with a microfluidics system capable of precise delivery, in terms of location, time and volume, of analyte to each of the addressable sites present on the chip.
In yet another embodiment, a device consisting of a chip with a microfluidics system used in combination with optical monitoring is disclosed. In this embodiment, the chip is comprised of separate addressable sites for the purposes of analyte separation, processing, and modification, and is used in conjunction with a microfluidics system capable of precise delivery of analyte to each of the addressable sites present on the chip, and is further used in combination with an optical monitoring, such as SPR, to track the precise location of the analyte and monitor progress throughout the analytical process.
In a further embodiment, a device for BCMS is disclosed. Preferably, a chip comprised of separate addressable sites for the purposes of analyte separation, processing and modification is used in conjunction with a microfluidics system capable of precise delivery of analyte to each of the addressable sites present on the chip. Optical monitoring, in the form of SPR, is used to track the precise location of the analyte and monitor progress throughout the analytical method, after which mass spectrometry, in the form of MALDI-TOF, is used to structurally characterize the analyte and modified analyte resulting from the analytical method.
In another embodiment, a method for performing the modification, or bioreaction, of biomolecules is disclosed. Preferably, the method involves capturing an analyte present within a sample by an interactive surface layer located in a separation site; washing unwanted portions of the sample from the surroundings of the captured analyte; transferring the captured analyte from the separation site to a modifying site; and modifying or bioreacting the analyte to create a modified or bioreacted analyte. The modified analyte may then be subsequently characterized and/or identified by techniques such as mass spectrometry.
In another embodiment, a method for performing activated bimolecular interaction analysis is disclosed. Preferably using surface plasmon resonance-mass spectroscopy on the sample. The method involves capturing an analyte present within the sample by an interactive surface layer of an IA sensor located in a separation site; washing unwanted biomolecules from the surroundings of the captured analyte; transferring the captured analyte from the separation site to a modifying site; bioreacting the analyte to create an modified or bioreacted analyte; analyzing the activated analyte by techniques such as surface plasmon resonance while the analyte is captured by an interactive surface layer of the bioactive chip BC located in either or both the separation and/or modifying sites; and, optionally, identifying the activated analyte by desorbing/ionizing the activated analyte from the interactive surface layer of the bioactive chip BC located on the modifying site while under vacuum within a mass spectrometer.
In yet another embodiment, a method for the analysis of multiple analytes present in a sample is disclosed. In this embodiment, a sample containing multiple analytes is brought in contact with an separation site present on the surface of a chip derivatized with a receptor. SPR is used to monitor the interaction between the immobilized receptor and the analytes, after which mass spectrometry, in the form of MALDI-TOF, is used to determine the number and nature of analytes retrieved by the receptor.
The novel features that are considered characteristic of the invention are set forth with particularity in the appended claims. The invention itself, however, both as to its structure and its operation together with the additional object and advantages thereof will best be understood from the following description of the preferred embodiment of the present invention when read in conjunction with the accompanying drawings. Unless specifically noted, it is intended that the words and phrases in the specification and claims be given the ordinary and accustomed meaning to those of ordinary skill in the applicable art or arts. If any other meaning is intended, the specification will specifically state that a special meaning is being applied to a word or phrase. Likewise, the use of the words xe2x80x9cfunctionxe2x80x9d or xe2x80x9cmeansxe2x80x9d in the Description of Preferred Embodiments is not intended to indicate a desire to invoke the special provision of 35 U.S.C. xc2xa7112, paragraph 6 to define the invention. To the contrary, if the provisions of 35 U.S.C. xc2xa7 112, paragraph 6, are sought to be invoked to define the invention(s), the claims will specifically state the phrases xe2x80x9cmeans forxe2x80x9d or xe2x80x9cstep forxe2x80x9d and a function, without also reciting in such phrases any structure, material, or act in support of the function. Even when the claims recite a xe2x80x9cmeans forxe2x80x9d or xe2x80x9cstep forxe2x80x9d performing a function, if they also recite any structure, material or acts in support of that means of step, then the intention is not to invoke the provisions of 35 U.S.C. xc2xa7112, paragraph 6. Moreover, even if the provisions of 35 U.S.C. xc2xa7112, paragraph 6, are invoked to define the inventions, it is intended that the inventions not be limited only to the specific structure, material or acts that are described in the preferred embodiments, but in addition, include any and all structures, materials or acts that perform the claimed function, along with any and all known or later-developed equivalent structures, materials or acts for performing the claimed function.